Phosphorylation of α-synuclein at T64 results in distinct oligomers and exerts toxicity in models of Parkinson's disease.

α-Synuclein accumulates in Lewy bodies, and this accumulation is a pathological hallmark of Parkinson's disease (PD). Previous studies have indicated a causal role of α-synuclein in the pathogenesis of PD. However, the molecular and cellular mechanisms of α-synuclein toxicity remain elusive. Here, we describe a novel phosphorylation site of α-synuclein at T64 and the detailed characteristics of this post-translational modification. T64 phosphorylation was enhanced in both PD models and human PD brains. T64D phosphomimetic mutation led to distinct oligomer formation, and the structure of the oligomer was similar to that of α-synuclein oligomer with A53T mutation. Such phosphomimetic mutation induced mitochondrial dysfunction, lysosomal disorder, and cell death in cells and neurodegeneration in vivo, indicating a pathogenic role of α-synuclein phosphorylation at T64 in PD.

GPATCH4 contributes to nucleolus morphology and its dysfunction impairs cell viability.

The nucleolus serves a multifaceted role encompassing not only rRNA transcription and ribosome synthesis, but also the intricate orchestration of cell cycle regulation and the modulation of cellular senescence. G-patch domain containing 4 (GPATCH4) stands as one among the nucleolar proteins; however, its functional significances remain still unclear. In order to elucidate the functions of GPATCH4, we examined the effects of its dysfunction on cellular proliferation, alterations in nucleolar architecture, apoptotic events, and cellular senescence. Through experimentation conducted on cultured neuroblastoma SH-SY5Y cells, the reduction of GPATCH4 caused inhibition of cellular proliferation, concurrently fostering escalated apoptotic susceptibilities upon exposure to high-dose etoposide. In the realm of nucleolar morphology comparisons, a discernible decline was noted in the count of nucleoli per nucleus, concomitant with a significant expansion in the area occupied by individual nucleoli. Upon induction of senescence prompted by low-dose etoposide, GPATCH4 knockdown resulted in decreased cell viability and increased expression of senescence-associated markers, namely senescence-associated ß-galactosidase (SA-ß-GAL) and p16. Furthermore, GPATCH4 dysfunction elicited alterations in the gene expression profile of the ribosomal system. In sum, our findings showed that GPATCH4 is a pivotal nucleolar protein that regulates nucleolar morphology and is correlated with cell viability.

Evaluation of Ectopic Mitochondrial DNA in HeLa Cells.

The presence of ectopic DNA in the cytoplasm induces inflammation and cell death. It has been widely reported that leakage of nuclear DNA into the cytoplasm can mainly be sensed by cyclic GMP-AMP synthase (cGAS). We recently reported that mitochondria-derived cytoplasmic double-stranded DNA (dsDNA) that has escaped lysosomal degradation induces significant cytotoxicity in cultured cells and in vivo. Cytoplasmic mitochondrial DNA is assumed to be involved in various diseases and disorders, and more and more papers have been published confirming this. On the other hand, the current method for evaluating mitochondrial DNA in the cytoplasm may not be quantitative. Here, we introduce in detail a method to evaluate ectopic mitochondrial DNA in cells. This method is useful in basic research as well as in the study of aging, Parkinson's disease, Alzheimer's disease, heart failure, autoimmune diseases, cancer, and other conditions.

Cerebrospinal fluid injection into adult zebrafish for disease research.

A modified method of cerebrospinal fluid injection was developed for the efficient and reliable administration of substances to the zebrafish central nervous system. The accuracy of this modified method was evaluated using Alexa Fluor dye injection. A high survival ratio was achieved due to the simplicity of the procedure and ice-tricaine combined anaesthesia. To validate this new method, we injected ammonium chloride, which successfully blocked lysosome function resulting in elevated LC3-II and the accumulation of ubiquitinated proteins. Injection of human α-synuclein fibrils initiated a prion-like propagation of α-synuclein pathology in zebrafish. This method can be used to investigate the effects of various substances and the propagation of α-synuclein in the central nervous system.

Enhancement of secondary xylem cell proliferation by Arabidopsis cyclin D overexpression in tobacco plants.

UNLABELLED: Secondary xylem is composed of daughter cells produced by the vascular cambium in the stem. Cell proliferation of the secondary xylem is the result of long-range cell division in the vascular cambium. Most xylem cells have a thickened secondary cell wall, representing a large amount of biomass storage. Therefore, regulation of cell division in the vascular cambium and differentiation into secondary xylem is important for biomass production. Cell division is regulated by cell cycle regulators. In this study, we confirm that cell cycle regulators influence cell division in the vascular cambium in tobacco. We produced transgenic tobacco that expresses Arabidopsis thaliana cyclin D2;1 (AtcycD2;1) and AtE2Fa-DPa under the control of the CaMV35S promoter. Each gene is a positive regulator of the cell cycle, and is known to influence the transition from G1 phase to S phase. AtcycD2;1-overexpressing tobacco had more secondary xylem cells when compared with control plants. In order to evaluate cell division activity in the vascular cambium, we prepared a Populus trichocarpa cycB1;1 (PtcycB1;1) promoter containing a destruction box motif for ubiquitination and a ß-glucuronidase-encoding gene (PtcycB1;1pro:GUS). In transgenic tobacco containing PtcycB1;1pro:GUS, GUS staining was specifically observed in meristem tissues, such as the root apical meristem and vascular cambium. In addition, mitosis-monitoring plants containing AtcycD2;1 had stronger GUS staining in the cambium when compared with control plants. Our results indicated that overexpression of AtcycD enhances cell division in the vascular cambium and increases secondary xylem differentiation in tobacco. KEY MESSAGE: We succeeded in inducing cell proliferation of cambium and enlargement of secondary xylem region by AtcycD overexpression. We also evaluated mitotic activity in cambium using cyclin-GUS fusion protein from poplar.

Loss of GBA in zebrafish leads to dopaminergic neurodegeneration, but overexpression of α-synuclein does not further worsen degeneration.

OBJECTIVES: Parkinson's disease is a neurodegenerative disorder that causes motor and nonmotor symptoms due to the loss of dopaminergic nerves and is characterized by the presence of Lewy bodies, which are mainly composed of α-synuclein. Glucosylceramidase beta (GBA), which is a causative gene of autosomal recessive Gaucher disease, is also known to be a risk gene for Parkinson's disease. In this study, we tried to detect synergistic effects of α-synuclein accumulation and gba depletion on dopaminergic neurodegeneration in zebrafish. METHODS: We generated a transgenic line of zebrafish overexpressing the A53T α-synuclein and gba mutant fish, and analyzed pathologies of α-synuclein aggregation and neurodegeneration. RESULTS: Zebrafish overexpressing the A53T α-synuclein did not exhibit α-synuclein aggregate formation. After the loss of gba function in this mutant α-synuclein transgenic line, we observed the marked presence of α-synuclein aggregates. Loss of gba function in zebrafish resulted in dopaminergic and noradrenergic neurodegeneration but this level of neurodegeneration was not exacerbated by overexpression of mutant α-synuclein. CONCLUSIONS: These results indicate that loss of gba function was sufficient to generate a neurodegenerative phenotype in zebrafish regardless of the expression of α-synuclein.

Cytosolic dsDNA of mitochondrial origin induces cytotoxicity and neurodegeneration in cellular and zebrafish models of Parkinson's disease.

Mitochondrial dysfunction and lysosomal dysfunction have been implicated in Parkinson's disease (PD), but the links between these dysfunctions in PD pathogenesis are still largely unknown. Here we report that cytosolic dsDNA of mitochondrial origin escaping from lysosomal degradation was shown to induce cytotoxicity in cultured cells and PD phenotypes in vivo. The depletion of PINK1, GBA and/or ATP13A2 causes increases in cytosolic dsDNA of mitochondrial origin and induces type I interferon (IFN) responses and cell death in cultured cell lines. These phenotypes are rescued by the overexpression of DNase II, a lysosomal DNase that degrades discarded mitochondrial DNA, or the depletion of IFI16, which acts as a sensor for cytosolic dsDNA of mitochondrial origin. Reducing the abundance of cytosolic dsDNA by overexpressing human DNase II ameliorates movement disorders and dopaminergic cell loss in gba mutant PD model zebrafish. Furthermore, IFI16 and cytosolic dsDNA puncta of mitochondrial origin accumulate in the brain of patients with PD. These results support a common causative role for the cytosolic leakage of mitochondrial DNA in PD pathogenesis.

Risk factors related to the development of pressure ulcers in the critical care setting.

AIM: This study identified risk factors for pressure ulcer development early in the admission stage to determine what appropriate interventions might be conducted early in the admission stage to decrease the risk of pressure ulcer development. BACKGROUND: Among patients who develop pressure ulcers, 8-40% of them occur in a critical care setting. Therefore, the development of pressure ulcers is a common problem. DESIGN: Prospective cohort study. METHODS: The study facility was a 606-bed tertiary care hospital. Ninety eight patients admitted to the intensive care unit (ICU) or the high care unit (HCU) from 16 April 2003-15 July 2003 were evaluated in the final analysis. RESULTS: The mean patient age was 62.3 (SD 16.1) years, and the incidence of pressure ulcers in this study was 11.2% during the observation period. Multivariate analysis showed that 'emergency ICU/HCU patients' and 'infrequent turning' were related to pressure ulcer development. Patients with pressure ulcers experienced significantly fewer turns and repositionings (OR = 0.452, 95% CI: 0.212-0.966], p < 0.05. Fewer pressure ulcers developed in scheduled ICU/HCU patients than in emergency ICU/HCU patients (OR = 0.041 [95% CI: 0.004-0.470], p < 0.01). CONCLUSION: There was no relationship between pressure ulcer development and APACHE II score, or any medication that affected skin integrity. The frequency of turning and repositioning and patients with an emergency admission to the ICU/HCU can be the prognostic indicators for developing scoring system in critical care settings. RELEVANCE TO CLINICAL PRACTICE: These patients admitted directly to ICU or HCU were in a high risk group, further preventive strategies will be required.

Degeneration of dopaminergic neurons and impaired intracellular trafficking in Atp13a2 deficient zebrafish.

ATP13A2 is the autosomal recessive causative gene for juvenile-onset Parkinson's disease (PARK9, Parkinson's disease 9), also known as Kufor-Rakeb syndrome. The disease is characterized by levodopa-responsive Parkinsonism, supranuclear gaze palsy, spasticity, and dementia. Previously, we have reported that Atp13a2 deficient medaka fish showed dopaminergic neurodegeneration and lysosomal dysfunction, indicating that lysosome-autophagy impairment might be one of the key pathogeneses of Parkinson's disease. Here, we established Atp13a2 deficient zebrafish using CRISPR/Cas9 gene editing. We found that the number of TH + neurons in the posterior tuberculum and the locus coeruleus significantly reduced (dopaminergic neurons, 64 % at 4 months and 37 % at 12 months, p < 0.001 and p < 0.05, respectively; norepinephrine neurons, 52 % at 4 months and 40 % at 12 months, p < 0.001 and p < 0.05, respectively) in Atp13a2 deficient zebrafish, proving the degeneration of dopaminergic neurons. In addition, we found the reduction (60 %, p < 0.05) of cathepsin D protein expression in Atp13a2 deficient zebrafish using immunoblot. Transmission electron microscopy analysis using middle diencephalon samples from Atp13a2 deficient zebrafish showed lysosome-like bodies with vesicle accumulation and fingerprint-like structures, suggesting lysosomal dysfunction. Furthermore, a significant reduction (p < 0.001) in protein expression annotated with vesicle fusion with Golgi apparatus in Atp13a2 deficient zebrafish by liquid-chromatography tandem mass spectrometry suggested intracellular trafficking impairment. Therefore, we concluded that Atp13a2 deficient zebrafish exhibited degeneration of dopaminergic neurons, lysosomal dysfunction and the possibility of intracellular trafficking impairment, which would be the key pathogenic mechanism underlying Parkinson's disease.

Comparative assessment of photodynamic therapy for typical age-related macular degeneration and polypoidal choroidal vasculopathy: a multicenter study in Hyogo prefecture, Japan.

PURPOSE: We aimed to evaluate the effects of photodynamic therapy (PDT) on different phenotypes of age-related macular degenerations (AMD): typical AMD (tAMD) and polypoidal choroidal vasculopathy (PCV). PROCEDURES: 246 eyes from 242 patients (tAMD: 139, PCV: 107 eyes) were recruited. Gender, age, best-corrected visual acuity (BCVA) before treatment, greatest linear dimension before treatment, lesion phenotype and PDT frequency were evaluated for predicting the BCVA at 12 months after PDT using stepwise multiple regression analyses. Additionally, 125 eyes with tAMD and 97 eyes with PCV followed up for more than 12 months after the final PDT were compared for the recurrence period. RESULTS: In the stepwise analysis, a younger age, better pretreatment BCVA, lower PDT frequency, lesions with PCV and a smaller pretreatment greatest linear dimension were all significantly beneficial for a better BCVA at 12 months after PDT. PCV showed a significantly lower PDT frequency and greater improvement in the BCVA than tAMD. The recurrence period of PCV was significantly later than that of tAMD. CONCLUSIONS: The phenotype of AMD is significantly correlated with its prognosis after PDT. PCV showed a significantly better response to PDT in terms of BCVA improvement and effect durability.

Continuous monitoring of interface pressure distribution in intensive care patients for pressure ulcer prevention.

AIM: This paper is a report of a study conducted to examine whether continuous interface pressure monitoring of postoperative patients in an intensive care unit is feasible in clinical practice. BACKGROUND: The interface pressure between skin and surfaces is generally evaluated for pressure ulcer prevention. However, the intensity and duration of interface pressure necessary for pressure ulcer development remains unclear because the conventional interface pressure sensors are unsuitable for continuous monitoring in clinical settings. METHODS: A total of 30 postoperative patients in an intensive care unit participated in this study in 2006-2007. A sensor was built into a thermoelastic polymer mattress. The whole-body interface pressure was recorded for up to 48 hours. Pressure ulcer development was observed during the morning bed-bath. For analysis, the intensity and duration of the maximal interface pressure was evaluated. FINDINGS: The mean age of the study group was 62.0 +/- 15.4 years. Two participants developed stage I pressure ulcer and blanchable redness at the sacrum. The longest duration of pressures greater than 100 mmHg were 487.0, 273.5 and 275.7 minutes in the pressure ulcer, blanchable redness and no redness groups respectively. CONCLUSION: Continuous monitoring of the intensity and duration of whole-body interface pressure using the KINOTEX sensor is feasible in intensive care patients.

Correlates of community health service utilization for menopausal symptoms among urban Japanese women.

OBJECTIVES: This study aims to identify the factors associated with community health service utilization for menopausal symptoms among Japanese women in urban areas by using Andersen's behavioral model of health service use. DESIGN: A cross-sectional study was conducted. SAMPLE: The sample comprised 350 women aged 40-60 years living in a community in Japan. MEASUREMENTS: The independent variables were predisposing (demographics, social status, and health beliefs), enabling (family and community resources), and need factors (perceived health status and the need for community health services). The dependent variable was community health service utilization. A logistic regression model was used. RESULTS: 8 of the women were categorized in "treatment required" by simplified menopausal index score, and 103 women said that they would use the services. Knowledge regarding the services, OR: 1.814 (1.084-3.036); the absence of a negative attitude toward menopausal syndromes, OR: 0.605 (0.367-0.998); and the presence of a positive attitude, OR: 2.203 (1.070-4.536); influenced community health service utilization. CONCLUSIONS: An appropriate attitude toward menopausal symptoms was observed to be more relevant to community health service utilization than the experience or acuity of the menopausal symptoms themselves. Therefore, community-dwelling, middle-aged women should be provided with sufficient education regarding menopausal symptoms to encourage their utilization of community health services.

Factors related to family visits to nursing home residents in Japan.

This study examined factors related to the frequency and duration of family visits to elderly residents of nursing homes in Japan. Data were collected using a self-administered questionnaire from 299 family members of residents of three nursing homes in Tokyo. Almost all family members visited residents more than once a month, and more than three quarters stayed for more than 1h during each visit. These findings confirm that family involvement with the elderly is maintained even after admission to a nursing home. Multiple regression analysis revealed the variables related to a higher frequency and duration of visits. Some factors related to frequent or long visits such as the requirement of higher care or the ability to remember family visits were similar to the findings of other studies conducted in western countries, yet the results need to be understood in a Japanese cultural context. Other factors such as the gender of the visitors, distance to the nursing home, and sentiment about placement were thought to be related to the unique cultural norms of family caregiving in Japan. These findings will be useful for developing effective methods to assist nursing home residents and their families in Japan.

[Case of metastatic intraocular malignant lymphoma with neovascular glaucoma].

BACKGROUND: It is relatively rare to encounter a case of neovascular glaucoma induced by malignant lymphoma metastasized into the eyeball. CASE: A 79-year-old woman initially visited our ophthalmology clinic with the chief complaint of blurring of vision in the left eye which was affected by neovascular glaucoma. She had a history of systemic malignant lymphoma, first diagnosed from skin biopsy and treated to complete remission 3 years previously. A metastatic brain lesion, detected 2 months before her initial visit to our clinic, was cured by radiotherapy. FINDINGS: By ultrasound biomicroscopy (UBM) we detected abnormal thickening of the temporal half of the ciliary body of her left eye. Cytological examination of the aqueous humor revealed invasion by malignant cells of presumed lymphocyte origin. Radiation therapy to the left eye normalized the intraocular pressure in a week, followed by a reduction in the neovascularization of the iris and the thickening of the ciliary body. CONCLUSION: This case showed that metastatic malignant lymphoma in the eye could result in neovascular glaucoma and that UBM is useful to detect and to observe lesions in the iris and ciliary body.

[Mental health and related factors with family caregivers for elderly in special-care nursing homes for the aged].

PURPOSE: The purpose of this study was to examine the mental health and related factors with family caregivers for the elderly in special-care nursing homes for the aged. METHODS: We conducted a questionnaire survey among family caregivers for elderly living in three special-care nursing homes in Tokyo. The questionnaire included factors from the General Health Questionnaire-28 (GHQ-28) pertaining to family caregivers, the elderly, and the relationships between them. The GHQ-28 was used to measure the mental health of the caregivers and a logistic regression model was applied for the analysis. RESULTS: Of the 145 family caregivers surveyed, 59 (40.7%) exhibited low mental health (GHQ-28> or = 7). The logistic regression analysis revealed that family caregivers with low mental health had lower social support (OR: 0.10 (0.03-0.29)) and had longer visiting times (OR: 5.80 (1.79-18.82)). DISCUSSION: The results suggested that many family caregivers for elderly persons in institutions may have poor mental health and that it is necessary to provide them with support. It is concluded that self-help groups for families and the promotion of communication between the elderly and their family caregivers might be effective for this purpose.

Optical coherence tomography detects characteristic retinal nerve fiber layer thickness corresponding to band atrophy of the optic discs.

OBJECTIVES: To analyze retinal nerve fiber layer (RNFL) thickness in eyes with band atrophy by use of optical coherence tomography (OCT) and to evaluate the ability of OCT to detect this characteristic pattern of RNFL loss. DESIGN: Cross-sectional, retrospective study. PARTICIPANTS: Thirty-four eyes of 18 patients with bitemporal hemianopia caused by optic chiasm compression by chiasmal tumors were studied. All eyes were divided into 3 groups according to visual field loss grading after Goldmann perimetry. INTERVENTIONS: Retinal nerve fiber layer thickness measurements with OCT. MAIN OUTCOME MEASURES: Retinal nerve fiber layer thickness around the optic disc was measured by OCT (3.4-mm diameter circle). Calculation of the changes in OCT parameters, including the horizontal (nasal + temporal quadrant RNFL thickness) and vertical values (superior + inferior quadrant RNFL thickness) was based on data from 160 normal eyes. Comparison between the 3 visual field grading groups was done with the analysis of variance test. The receiver operating characteristic (ROC) curve for the horizontal and vertical value were calculated, and the areas under the curve (AUC) were compared. RESULTS: Retinal nerve fiber layer thickness in eyes with band atrophy decreased in all OCT parameters. The reduction rate in average and temporal RNFL thickness and horizontal value was correlated with visual field grading. The AUC of horizontal value was 0.970+/-0.011, which was significantly different from AUC of vertical value (0.903+/-0.022). CONCLUSIONS: The degree of RNFL thickness reduction correlated with that of visual field defects. Optical coherence tomography was able to identify the characteristic pattern of RNFL loss in these eyes.

Goniosynechialysis with lens aspiration and posterior chamber intraocular lens implantation for glaucoma in spherophakia.

A 17-year-old girl presented with bilateral angle-closure glaucoma associated with spherophakia. A previous bilateral laser iridotomy failed to control intraocular pressure (IOP). Goniosynechialysis with lens aspiration and posterior chamber intraocular lens implantation were performed in both eyes. Peripheral iridoplasty was performed 3 days later. The postoperative IOP was controlled without medication for 12 months in the right eye and 24 months in the left eye. By restructuring the physiologic aqueous outflow route, goniosynechialysis safely and effectively treated secondary glaucoma from spherophakia.

Use of a designed Peptide library to screen for binders to a particular DNA g-quadruplex sequence.

We demonstrated a method to screen for binders to a particular G-quadruplex sequence using easily designed short peptides consisting of naturally occurring amino acids and mining of binding data using statistical methods such as hierarchical clustering analysis (HCA). Despite the small size of the library used in this study, candidates of specific binders were identified. In addition, a selected peptide stabilized the G-quadruplex structure of a DNA oligonucleotide derived from the promoter region of the protooncogene c-MYC. This study illustrates how a peptide library can be designed and presents a screening guideline for construction of G-quadruplex binders. Such G-quadruplex peptide binders could be functionally modified to enable switching, cellular penetration, and organelle-targeting for cell and tissue engineering.

Validation and determination of the sensing area of the KINOTEX sensor as part of development of a new mattress with an interface pressure-sensing system.

The purpose of the present study was to examine the validity of the KINOTEX sensor via comparison with an existing sensor and to determine the sensing areas for a new alternating-air mattress that incorporates an interface pressure-sensing system. The study design was an evaluation study to validate and determine the sensing area of the KINOTEX sensor in comparison with another sensor. Study participants were fifty-one healthy volunteers over eighteen years of age, and the two sensors were placed between participants and an alternating-air mattress. We measured the contact area, full weight load, and maximum pressure in the calcaneal region using two sensors and obtained a graphic pressure distribution of >40 mmHg in the lateral and supine positions. Correlation coefficients between sensors were r = 0.88 (p < 0.001) for the contact area, r = 0.89 (p < 0.001) for full weight load, and r = 0.72 (p < 0.001) at maximum pressure in the calcaneal region. Ninety-one percent of the pressure distribution was recorded in the central 50 cm of the bed, and 94.6% was recorded within an area 160 cm in length need the foot of the bed. We investigated the correlation between the KINOTEX sensor and an existing sensor and determined the necessary sensing area. Results suggested the feasibility of developing a new alternating-air mattress incorporating an interface pressure-sensing system to help prevent pressure ulcers.

Effect of vibration on skin blood flow in an in vivo microcirculatory model.

The effect of vibration on skin microcirculation was studied to investigate the possibility of clinical use of vibration to prevent and treat pressure ulcers. Vibrations at a vibrational intensity of 600, 800, or 1,000 mVpp with a fixed frequency of 47 Hz were applied horizontally to the ear of male hairless mice (n = 6 for each group) under inhalation anesthesia. The control group (n = 6) received no vibrations. Venular blood flow was measured by an intravital videomicroscope at the baseline and at 0, 5, and 15 min after the application of vibrations. A significant increase was observed in the 600 mVpp group 5 and 15 min after vibration in comparison to the control group (P = 0.002 and P = 0.046, respectively). We also detected increased blood flow in the 800 mVpp group (P = 0.028) and the 1,000 mVpp group (P = 0.012) 5 min after vibration; however, these increases attenuated after 15 min. These results indicate that direct skin vibration at a frequency of 47 Hz improves skin blood flow. The present study gives further support to the role of vibration on a short-term increase in skin blood flow.